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cmi12170-sup-0001-fs1.tif863K

Fig. S1. C3b deposition on all tested Gram-positive bacteria. Different Gram-positive bacteria were incubated with buffer, 10% normal human serum (NHS) or 10% heat-inactivated (HI) serum. C3b deposition was detected by using the monoclonal WM-1 antibody (recognizing the C3c part of C3b/C3bi) and flow cytometry. Data represent the mean fluorescence ± standard error of the mean (SEM) of three independent experiments.

cmi12170-sup-0002-fs2.tif1310K

Fig. S2. Dose-dependent C5b-9 deposition on all tested Gram-positive bacteria. Different Gram-positive bacteria were incubated with various concentrations of normal human serum in presence of buffer (−), 5 mM EDTA (inhibits all complement routes) or Ornithodoros moubata Complement Inhibitor (OmCI, 2 μg ml−1) (inhibits C5 proteolysis). Deposition of C5b-9 was determined using flow cytometry.

cmi12170-sup-0003-fs3.tif787K

Fig. S3. C5b-9 deposition is inhibited by SIC and SSL7. L. lactis was incubated with various concentrations of normal human serum in presence of buffer (−), 40 μg ml−1 SIC or 40 μg ml−1 SSL7. Deposition of C5b-9 was determined using flow cytometry. Data represent the mean fluorescence ± standard error of the mean (SEM) of three independent experiments.

cmi12170-sup-0004-fs4.tif1128K

Fig. S4. C5b-9 deposition on bacteria does not influence phagocytosis and killing by human neutrophils.

A. C5b-9 deposition on L. lactis in buffer, 10% NHS, 10% ΔC6 serum and 10% ΔC6 serum supplemented with 20 μg ml−1 C6.

B. Phagocytosis by human neutrophils of FITC-labelled L. lactis pre-incubated in buffer, 10% NHS, 10% ΔC6 serum or 10% ΔC6 serum supplemented with 20 μg ml−1 C6. Mean fluorescence of neutrophils is expressed as a measure for phagocytosis.

C. Killing of L. lactis by human neutrophils in presence of HI, 10% NHS, 10% ΔC6 serum and 10% ΔC6 serum supplemented with 20 μg ml−1 C6. Results are expressed as percentage surviving bacteria compared with the initial inoculum.

A–C. Data represent the mean fluorescence ± standard error of the mean (SEM) of three independent experiments.

cmi12170-sup-0005-fs5.tif1487K

Fig. S5. C5b-9 deposition requires complex formation at the bacterial surface. C5b-9 deposition on different Gram-positive bacteria after two-step incubations. First, bacteria were incubated with buffer, 10% NHS, purified C5b-9 (20 μg ml−1 C5b6, C7, C8 and C9) or 10% C5 depleted serum (ΔC5). After washing, bacteria were subsequently incubated with buffer or purified C5–9 (20 μg ml−1 purified C5, C6, C7, C8 and C9). Deposition of C5b-9 was determined by flow cytometry.

cmi12170-sup-0006-fs6.tif5398K

Fig. S6. C3b and C5b-9 deposition on Gram-positive bacteria. Related to Fig. 5, confocal images showing random distribution of C3b deposition and specific localization of C5b-9 on S. pyogenes, L. lactis, B. subtilis and S. aureus. Complement components are indicated in green and the lypophilic membrane dye in red.

cmi12170-sup-0007-fs7.tif3870K

Fig. S7. The entire C5b-9 complex specifically localizes on Gram-positive bacteria. Bacteria were incubated with 10% serum and C6 and C8 were detected using monoclonal antibodies that were stained using Alexa Fluor488-conjugated antibodies (green). Bacterial membranes were stained using the lypophilic dye FM5-95 (red). Representative images for three independent experiments on S. pyogenes, S. aureus, B. subtilis and L. lactis are shown.

cmi12170-sup-0008-si.doc56K

Supplementary experimental procedures.

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